Heterosexual transmission is the dominant mode of HIV-1 acquisition woridwide. Understanding the eariy innate immune response in the mucosa is, thus, essential for devising novel strategies to prevent infection. Compelling evidence suggests that immunological events occurring during the first days and weeks after HIV-1 infection are critical determinants shaping the course of HIV-1/AIDS disease. The mechanisms that underiie the failure of innate immune responses to restrict HIV-1 infection are currently under intense investigation. The goal of this program project is to dissect the early events in the innate immune response directed at HlV-1 using a systems biology approach. Our proposal (project 4) will use a primary DC-T cell system to test the hypothesis that HIV-1 manipulates the kinetics of human innate immune responses by interfering with dendritic cell (DC) function, in particular, the induction of type I interferon (IFN) in those cells. We speculate that HIV-1 delays DC maturation and that one or more of the HIV-1 proteins encodes an IFN antagonist. In specific aim #1 we will determine the reciprocal impact of HIV-1 infection and IFN/pattern recognition response signaling on each other. In specific aim #2 we will evaluate the role of newly identified cellular restriction factors in DCs within the context of viral infection. We will assess the efficiency of viral replication and transfer from dendritic cells to T lymphocytes, the pattern of DC activation and the IFN/PRR signaling pathways. We will use lentiviral transduction systems to down-regulate or over-express selected host factors (50-100) to test the effect of their gain or loss of function in myeloid DC lineages and T lymphocytes. In specific aim #3 we will identify putative viral inhibitors of DC maturation and IFN production using a series of primary viral isolates of different subtypes, HIV-1 full-length molecular clones deleted of accessory genes. We will confirm and expand our findings by inserting single accessory and regulatory HIV-1 genes into recombinant Newcastle Disease Virus (NDV) vectors which induce rapid and strong innate immune responses. This project combines the complementary areas of expertise of Dr. Ana Fernandez- Sesma and Dr. Viviana Simon. Dr. Fernandez-Sesma has extensive experience with primary human DCs and the initiation of immune responses in those cells by different viruses, such as Influenza, Dengue (DENV) and NDV. Dr. Simon has great expertise in HIV-1 molecular virology and host factors influencing HlV-1 replication, such as AP0BEC3. The results of our project will serve as raw data forthe mathematical models generated in project 6. This research project will determine the role of the restriction factors identified in project 1 and 2 on signaling, DC maturation, innate immune responses and viral inhibition in DCs and Tlymphocytes, both primary cell populations most relevant to mucosal immunity.